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標題: 人類胚胎幹細胞與胰蛋白&;#37238;篩選之人類胚胎幹細胞亞群間基因表現差異
Differential Expression Between The Human Embryonic Stem Cell And Trypsin-selected Human Embryonic Stem Cell Subpopulation
作者: 丁筱茜
Ting, Hsiao-Chien
關鍵字: Human Embyronic Stem Cells;人類胚胎幹細胞
出版社: 生命科學系所
人類胚胎幹細胞(human embryonic stem cells, HES cells)的單細胞繼代存活率很低,目前繼代人類胚胎幹細胞皆以細胞團塊的方式繼代以避免單細胞分離的情形。由於同質的(homogenous)和單細胞的人類胚胎幹細胞的產生相當困難,因此降低了人類胚胎幹細胞的轉染、clonal isolation與分化的效率。前人研究指出Y-27632可促進單細胞分離之人類胚胎幹細胞存活,但是其機制仍然不明。為了確定控制人類胚胎幹細胞的單細胞存活機制,本實驗室以單細胞繼代方式建立一株單細胞存活率較高的trypsin-resistant人類胚胎幹細胞亞群(sublines),期望找到trypsin-resistant人類胚胎幹細胞單細胞存活率較高的原因。為了了解trypsin-resistant與正常人類胚胎幹細胞的差異,本研究以上述兩種人類胚胎幹細胞為材料進行基因表現差異的分析。實驗結果發現細胞骨架材料(beta-actin、alpha-tubulin與beta-tubulin)與幫助細胞骨架生成的相關基因(ADD2、PAK3與FGD1)在trypsin-resistant人類胚胎幹細胞裡都有表現量提升的情形。因此假設由Rho/Rac/Cdc42訊息傳遞路徑活化肌動蛋白(actin-filament)與微管(microtubule)的穩定與增生可以幫助trypsin-resistant人類胚胎幹細胞抵抗單細胞分離逆境而存活。研究中也發現幫助肌動蛋白生成的表皮生長因子(epidermal growth factor, EGF)可以幫助單細胞的人類胚胎幹細胞存活。然而,細胞骨架的增生與穩定是否在人類胚胎幹細胞的單細胞存活扮演重要角色仍然需要驗證。未來會以微管控制劑(Taxol與nocodazol)驗證微管的穩定與人類胚胎幹細胞單細胞存活率的關係。本研究發現細胞骨架的穩定與增生可能增加人類胚胎幹細胞的單細胞存活率,期望可以解開人類胚胎幹細胞的單細胞存活機制。細胞骨架的穩定與強化在研究上或是醫學應用上或許是一種簡單、有效率且可以幫助產生同質的人類胚胎幹細胞株與提高人類胚胎幹細胞轉染效率的方法。

Human embryonic stem cell (HES cells) is highly sensitive to dissociation, which cause HES cells death. So far, the usual way to passage HES cells is using type IV collagenase or accutase to prevent dissociation of HES cells. Generation of homogenous and single HESC is an obstacle, preventing the efficient gene transfer and clonal isolation. Previous study demonstrates Y-27632 permit survival of dissociated HES cells, but the mechanism is still unclear. In order to find out the mechanism that regulate dissociated HES cells survival, trypsin-resistant HES sublines were obtained to find out what happended in trypsin-resistant HES sublines can let it survival from trypsin-dissociation. To explore the differences in trypsin-resistant HES cells and normal HES cells, the gene expression patterns of both HES cells are analyzed at protein- and RNA-level. We have found out that cytoskeleton materials (beta-actin, alpha-tubulin and beta-tubulin) and genes that promoting cytoskeleton formation (ADD2, PAK3 and FGD1) are up-regulate in trypsin-resistant HES cells. We postulate that the polymerization and stabilization of actin-filaments and microtubules by activation of Rho/Rac/Cdc42 pathway may help trypsin-resistant HES cells overcome dissociation and survive. Moreover, we also find that the epidermal growth factor (EGF) which induces actin-filament formation also increases the survival of dissociated HES cells. However, the relationship between cytoskeletons and HES cell survival still need to be confirmed. We will further treat HES cells with microtubule regulator (Taxol and nocodazol) to confirm the relationship between microtubule and HES cell survival. Since we have find that actin-filaments and microtubules may improve survival of trypsin dissociated HES cells, the survival mechanism of dissociated HES cells may be solve. With the enhancement of cytoskeleton, it may be a simple and more efficient process to generate homogenous population of HES cells and improve gene transfer efficiency of HES cells in medical research and applications.
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